Phenyl-alpha-tert-butyl nitrone reverses mitochondrial decay in acute Chagas' disease

Abstract

In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas' disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl-alpha-tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (>or=40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas' disease.

Figure 1

Myocardial respiratory complex activities in T. cruzi-infected (±PBN) mice. Shown are the specific activities of CI (A) and CIII (B) respiratory complexes and citrate synthase (C) in cardiac mitochondria of normal (N), infected/untreated (IUT), and infected/PBN-treated (IPBNT) mice, measured by spectrophotometric methods. Histochemical staining. Respiratory complexes of cardiac mitochondria were resolved by BN-PAGE. The BN gels were processed by either staining the whole gel with Coomassie blue (F) or subjecting gel slices containing individual complexes to enzymatic colorimetric reactions (D). Densitometric quantitation of catalytic staining for each respiratory complex was normalized to a Coomassie-stained complex (E). The data (mean ± SD) are representative of three independent experiments (n = 3 per experiment/group). **^##P < 0.01; *^#P < 0.05.

Figure 2

ATP content in T. cruzi-infected (±PBN) mice. Shown is the ATP content present in heart homogenates (A) and freshly isolated cardiac mitochondria (B) from normal (N), infected/untreated (IUT), and infected/PBN-treated (IPBNT) mice. H₂O₂ level in cardiac mitochondria of infected (±PBN) mice. C: Freshly isolated cardiac mitochondria were used to evaluate total H₂O₂ levels. D: Mitochondrial H₂O₂ release rate was measured in presence of complex II substrate (succinate). Data (mean ± SD) are representative of three independent experiments (n = 3 mice/experiment/group). **^##P < 0.01; *^#P < 0.05.

Figure 3

Transmission electron microscopy of heart tissue from T. cruzi-infected (±PBN) mice. A: Representative micrographs from ultrathin sections of heart tissue obtained from normal (N), infected/untreated (IUT), and infected/PBN-treated (IPBNT) mice are shown. Swollen mitochondria with extensive loss of cristae are presented in IUT. Note the absence of these features in cardiac myocyte of N and IPBNT mice. Oxidative modifications of cardiac mitochondrial membranes in infected (±PBN) mice. B: Lipid peroxidation products, ie, malonyldialdehydes, in cardiac mitochondria of acutely infected and infected/PBN-treated mice were compared by a thiobarbituric acid-reactive substance assay. C: Isolated cardiac mitochondria were derivatized with dinitrophenylhydrazide, and the DNP-reactive proteins identified by Western blot analysis. D: The results of Coomassie blue staining of the membrane used for Western blotting is shown. Data are representative of five independent experiments (n = 3 mice/group). **^##P < 0.01. Scale bar = 1 μm. Original magnifications, ×14,100.

Figure 4

mtRNA in T. cruzi-infected (±PBN) murine hearts. Northern blotting. Cardiac total RNA was resolved by denaturing agarose gel electrophoresis and then transferred to a nylon membrane. A: Northern blot analysis was performed with ³²P-labeled, mitochondria-specific cDNA probes. Phosphorimages of mitochondria-encoded transcripts were quantitated by densitometric analysis and normalized to the amounts of 18S rRNA. Real-time RT-PCR. Total RNA was reverse-transcribed to synthesize first-strand cDNA and subsequently used as a template with gene-specific primer pairs. B: Results were normalized to GAPDH. 1/2^Ct values, representative of mRNA expression levels are plotted on the y axis. The SD for all of the data points was <5%. Data are representative of two independent experiments (n = 3 mice/experiment/group). *^#P < 0.05.

Figure 5

Carbonylation of respiratory complex subunits in cardiac mitochondria of infected (±PBN) mice. Myocardial mitochondria were solubilized and the subunits of CI, CIII, and CIV respiratory chain complexes resolved by two-dimensional BN-PAGE. A–C: The oxidatively modified proteins were detected by immunoblotting with anti-DNP antibody. D–F: Coomassie blue stains of the membranes used for immunoblot analysis are shown. Data are representative of two independent experiments (n = 3 mice/group).

Figure 6

Quantitation of parasite burden. A: T. cruzi rDNA-specific PCR was performed for 28 cycles using total DNA isolated from whole heart tissue of normal (N), infected (IUT), and infected/PBN-treated (IPBNT) mice. B: Densitometric quantification of the signal from PCR amplification of T. cruzi in heart tissues was normalized to mouse β-actin. Histological analysis. Heart sections were subjected to H&E staining and visualized by light microscopy. C: Shown are representative micrographs from three independent experiments. Arrows represent parasite foci. H₂O₂ level. D: Freshly prepared cardiac homogenates were used to evaluate total H₂O₂ levels. Data (mean ± SD) are representative of three independent experiments (n = 3 mice/experiment/group). **^##P < 0.01. Original magnifications, ×20.